Like embryonic stem cells (ESCs), induced pluripotent stem (iPS) cells are able to differentiate into every cell type in the body, but their derivation (known as reprogramming) is more straightforward technically, and can be performed without the controversial use of donated eggs or embryos. The recent discovery of methods to reprogram human adult cells into iPS cells has generated much excitement due to the potential of iPS cells for patient-specific regenerative medicine, as well as for the new opportunities made possible in the study of embryonic development and cellular differentiation, and for the ability to create disease-specific cell lines for drug testing and the study of disease mechanism. Current research efforts are focused on 1) finding improved methods for iPS cell reprogramming, because current protocols are slow, highly inefficient, and possibly oncogenic, 2) directing the differentiation of iPS cells into the various cell types of the body, and 3) determining the safety of iPS cells for regenerative therapy. Until now, the study of iPS cell reprogramming and differentiation has focused on changes in gene transcription, DNA methylation, and histone modification, but surprisingly little attention has been devoted to cellular signaling by protein phosphorylation, even though this is the most prevalent signaling mechanism in eukaryotic cells. In order to illuminate the "black box" of kinase signaling during reprogramming and differentiation, we are developing methods to monitor protein phosphorylation in iPS cells. Because iPS reprogramming and differentiation occur in heterogeneous cell populations, the use of flow cytometry to obtain single cell measurements is crucial to identify the rare signaling populations of interest. Using phospho-specific flow cytometry protocols developed in the Nolan laboratory, and the reprogramming expertise of our collaborator Marius Wernig, we will characterize the differences in kinase signaling between pluripotent and differentiated cell types, and then monitor kinase signaling during iPS cell reprogramming. The identification of key signaling events will guide our efforts to identify conditions that increase the speed, efficiency, and safety of iPS cell reprogramming. In addition to providing unprecedented insight into the reprogramming process, we will also lay the groundwork for studying kinase signaling during differentiation of iPS cells and ESCs into any of the ~200 cell types of the body. Studying the differentiation of every cell type is beyond the scope of this proposal, but as a first step we will identify the kinase signaling events required for ESC/iPS cell differentiation into Hematopoietic Stem Cells (HSCs), a process that holds great promise for human therapy because it provides a renewable source of patient-matched HSCs for treating hematologic disease and for generating blood in vitro. Identifying the key signaling events in HSC derivation will guide efforts to find improved differentiation protocols, and single cell analysis by flow cytometry will be an important clinical tool to monitor the purity and safety of iPS cell-derived HSCs for regenerative medicine. PUBLIC HEALTH RELEVANCE: Like embryonic stem cells (ESCs), induced pluripotent stem (iPS) cells can differentiate into every cell type in the body, but their derivation (known as reprogramming) is more straightforward technically, and can be performed without the controversial use of donated eggs or embryos. iPS cells hold great promise for regenerative medicine, and for the study of disease mechanisms and human development, but the cellular signaling that controls their reprogramming and differentiation remains poorly understood. We are developing methods to measure protein phosphorylation (the most common mechanism of cellular signaling) in iPS cells, and we will use the key signaling events we identify to improve the speed and efficiency of iPS derivation, as well as the safety and utility of iPS cells for regenerative medicine.